The present invention relates generally to a method for treatment of xcex2-hemoglobinopathies. More specifically, this invention relates to the treatment of xcex2-hemoglobinopathies by administering a composition that promotes an increase in the relative amount of fetal erythropoiesis.
Normal adult hemoglobin comprises four globin proteins, two of which are alpha (xcex1) proteins and two of which are beta (xcex2) proteins. During fetal development in mammals (particularly in humans) the fetus produces a fetal hemoglobin which comprises two gamma (xcex3)-globin proteins instead of the two xcex2-globin proteins. At some point during fetal development or infancy, depending on the particular species and individual, there is a globin switch wherein the erythrocytes in the fetus switch from making predominantly xcex3-globin to making predominantly xcex2-globin. The developmental switch from production of predominantly fetal hemoglobin (HbF, xcex12xcex32) to production of adult hemoglobin (HbA, xcex12xcex22) occurs beginning at about 28 to 34 weeks of gestation and continues shortly after birth until HbA becomes predominant. This switch results primarily from decreased transcription of the gamma-globin genes and increased transcription of beta-globin genes. The blood of a normal adult contains only about 2% HbF.
Peripheral blood contains clonogenic cells that produce erythroid colonies and bursts in semisolid culture, given the appropriate combination of growth factors. Individual cells in such colonies can accumulate fetal hemoglobin (HbF), adult hemoglobin (HbA) or a combination of both. The pattern of hemoglobin expression and accumulation is different in cultures from fetal and adult blood. In cultures from adult blood, nucleated red cells accumulate either HbA (Fxe2x88x92A+) only or a combination of HbF and HbA (F+A+). Papayannopoulou, et al., Science 199: 1349-1350 (1978); Migliaccio, et al., Blood 76: 1150-1157 (1990). Individual colonies contain both F+ and Fxe2x88x92 cells, indicating that both types are progeny from the same circulating stem cells. Thus, during the early stages of development in culture, cells execute an option whether or not to express HbF. The proportion of adult F+ cells developing in culture does not appear to be preprogrammed in vivo, but appears to depend on culture conditions: A shift into the combined HbF and HbA expression pathway can, for example, be achieved in vitro by high serum concentrations, due to the activity of an unidentified compound that can be absorbed on activated charcoal. Bohmer, et al., Prenatal Diagnosis 19: 628-636 (1999); Migliaccio, et al., Blood 76: 1150 (1990); Rosenblum, et al., in: Experimental Approaches for the Study of Hemoglobin 397 (1985).
Hemoglobinopathies encompass a number of anemias of genetic origin in which there is a decreased production and/or increased destruction (hemolysis) of red blood cells (RBCs). There are genetic defects that result in the production by the body of abnormal hemoglobins with a concomitant impaired ability to maintain oxygen concentration. Some such disorders involve the failure to produce normal xcex2-globin in sufficient amounts, some involve the failure to produce normal xcex2-globin entirely. These disorders associated with the xcex2-globin protein are referred to generally as xcex2-hemoglobinopathies. For example, xcex2-thalassemias result from a partial or complete defect in the expression of the xcex2-globin gene, leading to deficient or absent HbA; sickle cell anemia results from a point mutation in the xcex2-globin structural gene, leading to the production of an abnormal (sickled) hemoglobin (HbS).
Sickle cell anemia (sickle cell disease, SCD) is an inherited, chronic, hemolytic anemia characterized by sickle-shaped RBCs. Because deoxygenated HbS is much less soluble than deoxy HbA, it forms a semisolid gel of rod-like tactoids, causing the RBCs to assume a sickle shape. HbS RBCs are more fragile than normal RBCs and hemolyze more readily, leading eventually to anemia.
It has been observed that certain populations of adult patients with beta chain abnormalities have higher than normal levels of fetal hemoglobin (HbF) and have been observed to have a milder clinical course of disease than patients with normal adult levels of HbF. For example, a group of Saudi Arabian sickle-cell anemia patients who express 20-30% HbF have only mild clinical manifestations of the disease. Pembrey, et al., Br. J. Haematol. 40: 415-429 (1978). There are also a variety of distinct genetic mutations that cause hereditary persistence of HbF, in which gamma-globin gene expression is not downregulated during development. This condition has been shown to significantly decrease the severity of sickle cell anemia or xcex2-thalassemia in individuals simultaneously affected with both traits. Wood and Weatherall, Biochem J. 215: 1-10 (1983). It is now accepted that hemoglobin disorders, such as sickle cell anemia and the xcex2-thalassemias, are ameliorated by increased HbF production. Reviewed in Jane and Cunningham Br. J. Haematol. 102: 415-422 (1998). See, also, Bunn, N. Engl. J. Med. 328: 129-131 (1993).
While the developmental switch from gammaxe2x80x94to betaxe2x80x94globin gene expression is strictly controlled, there is evidence that external factors can influence gamma-globin gene expression. For example, a delay in the fetal to adult hemoglobin switch has been observed in infants of diabetic mothers, suggesting an affect by circulating physiological factors. Perrine, et al., N. Engl. J. Med. 312: 334-338 (1985). Additionally, the ability to enhance HbF synthesis in vivo by pharmacological manipulation was demonstrated in baboons treated with 5-azacytidine (5-AzaC). DeSimone, et al., Proc. Natl. Acad. Sci, USA 79: 4428-4431 (1982). Subsequent studies confirmed the ability of 5-AzaC to increase HbF in patients with xcex2-thalassemia and sickle cell disease. Ley, et al., N. Engl. J. Medicine, 307: 1469-1475 (1982), and Ley, et al., Blood 62: 370-380 (1983).
Other agents that stimulate HbF in vivo include hydroxyurea [Carache, et al., N. Engl. J. Med. 332: 1317-1322 (1995)], butyrates [Perrine, et al., N. Engl. J. Med. 328: 81-86 (1993); Perrine, et al., Am. J. Pediatr. Hematol. Oncol. 16: 67-71 (1994)], activin and inhibin (U.S. Pat. No. 4,997,815), and various organic acids (e.g. valeric, polyhydroxy-benzoic, phenylacetic, mandelic) See, e.g., U.S. Pat. Nos. 5,366,996 and 5,700,640. Although these agents act via mechanisms that are not yet completely understood, it is thought that they partially derepress gamma-globin gene expression, leading to increased levels of HbF.
The effectiveness of many of these therapeutic agents has been demonstrated in several clinical trials, but is limited by unwanted side effects and variability in patient responses. Jane and Cunningham, Br. J. Haematol. 102: 415-422 (1998); Olivieri, Seminars in Hematology 33: 24-42 (1996). For example, very high dosages of butyric acid are necessary for inducing gamma-globin gene expression, requiring catheritization for continuous infusion of the compound. Moreover, these high dosages of butyric acid can be associated with neurotoxicity and multiorgan damage. Blau, et al., Blood 81: 529-537 (1993). There are also limitations to the therapeutic use of hydroxyurea; potential long-term consequences of treatment with this compound include teratogenic and oncogenic effects. While even minimal increases in HbF levels are helpful in sickle cell disease, xcex2-thalassemias require a much higher increase that is not reliably, or safely, achieved by any of the currently used agents. Olivieri, Seminars in Hematology 33: 24-42 (1996).
Thus, a need remains in the art for additional, novel, therapeutic methods for treatment of xcex2-hemoglobinopathiesxe2x80x94with reduced toxicityxe2x80x94capable of sustained induction of HbF.
The present invention provides a method for ameliorating xcex2-globin disorders in a mammal. In one aspect of the invention, the treatment involves ex vivo treatment of early erythroid progenitor cells that leads to an increase in the relative amounts of cells subsequently expressing and accumulating HbF. The cell treatment is to be followed by transplantation of the modified cells. In another aspect of the invention, the same modification of progenitor cells occurs in vivo. Both treatments are based on the novel discovery that the modification can be performed very early in the erythroid maturation process, without disturbance of the subsequent proliferation and maturation of the erythrocyte. The present invention also provides a procedure for the monitoring of xcex2-globinopathies and the response of a patient to treatment. In this aspect of the invention, erythropoiesis of a patient is studied (in vivo or in vitro) by generating profiles of correlated contents of different types of hemoglobin present in nucleated red cells (e.g. HbA vs. HbF, HbF vs. HbS, or HbS vs. HbA profiles).